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Isolation and Culture of Human Airway Smooth Muscle Cells

Airway smooth muscle (ASM) cells may be considered an important modulator of airway inflammatory responses, since they synthesize cytokines, chemokines, growth factors and metalloproteinases as well as support the adhesion of activated T lymphocytes. The study of the cellular and m ...

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Isolation and Purification of Human Mast Cells and Basophils

Mast cells were once thought to represent a single population of highly granulated secretory cells. However, with the development of mast cell isolation techniques for a diverse range of tissues, it became apparent that mast cells from different species and those from different tissues wi ...

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Macrophages: Identification, Separation, and Function

Within the human lung, macrophages can be found in the pleura, interstitium, alveoli, airways, vasculature, and walls of the bronchi and bronchiols. This distribution does not simply reflect the ubiquitous nature of these cells, as the macrophages found at these different sites show subtle d ...

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Neutrophils: Collection, Separation, and Activation

Neutrophils are manufactured in the bone marrow from stem cell precursors (myeloblasts) by a series of cell divisions. In healthy individuals only the mature neutrophils enter peripheral blood. They are the most numerous leukocyte subgroup, constituting half of the circulating white ...

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Collection, Separation, and Activation of Human T Lymphocytes

There is increasing evidence that T lymphocytes play a central role in regulating both the initial and chronic inflammatory cascades of allergic asthma (1,2) and can also regulate baseline airway responsiveness in mouse models of asthma (3). In particular, activation of antigen-specif ...

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Eosinophils: Collection, Separation, and Activation

Granulocytes play an important role in the host defense against invading microorganisms such as bacteria and fungi (1,2). For the killing reaction, these cells have an extensive machinery of cytotoxic effector mechanisms including phagocytosis, production of toxic oxygen metabo ...

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Analysis of Gene Expression: Reverse Transcription-Polymerase Chain Reaction

Numerous techniques have been developed to measure gene expression in tissues and cells. These include coupled reverse transcription and polymerase chain reaction amplification (RT-PCR), Northern blot (see Chapter 20), in situ hybridization (see Chapter 21), RNase protection as ...

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Analysis of Gene Expression: Northern Blotting

Messenger (m)RNAs represent the information-carrying intermediates for protein synthesis. Northern blotting gives a snapshot of the relative abundance of an RNA transcript at a set time point by measuring the steady-state levels of an RNA transcript. The steady-state level of a partic ...

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The Technique of In Situ Hybridization

In situ hybridization (ISH) (also called “hybridization histochemistry” or “hybridization cytology”) was first described in 1969 by Gall and Pardue who used the technique to localize ribosomal DNA in Xenopus oocytes (1). In contrast to other techniques of hybridization (i.e., Northern ...

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Measurement of Airway Mucin Gene Expression

Hypersecretion of airway mucus is characteristic of several severe lung diseases, particularly those involving chronic inflammation such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) (1). Mucins are the major macromolecular component of mu ...

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Transcription Factors and Inflammatory Gene Regulation: Strategic Approaches

In chronic inflammatory diseases, the expression of multiple genes, including those for cytokines, chemokines, adhesion molecules, receptors, and inflammatory enzymes, is often upregulated. The problem for many academic or industrial scientists is to elucidate the mechanisms ...

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Flow Cytometry: Measurement of Leukocyte Receptor Expression and Function

Flow cytometry is an invaluable tool for the analysis of leukocyte populations in inflammation. Flow cytometers, particularly those designed purely for analysis rather than cell sorting, have now become user-friendly machines and are commonplace in many laboratories. Any cell type ...

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Inflammatory Mediators in Spontaneously Produced Sputum

Airway inflammation is currently the subject of intense research interest concerning both the nature of the inflammatory cells, proteins, and cytokines present. These data are being used to define and assess the severity, cause, prognosis, and response to treatment of airway inflammat ...

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Measurement of Granulocyte Pharmacodynamics in Whole Blood by Flow Cytometry

During the Phase I/II assessment of new therapies with the potential to suppress eosinophil and neutrophil inflammation, there is a need to assess the peripheral blood pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the drug. This has relevance in respiratory disease since dr ...

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Current In Vitro Models of Leukocyte Migration: Methods and Interpretation

A large component of airway inflammatory disease is the recruitment of activated leukocytes (primarily eosinophils and T lymphocytes) from the lung vasculature into the bronchial walls resulting in lung edema. Ultimately, many of the infiltrating leukocytes progress across the a ...

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Tracing Intracellular Mediator Storage and Mobilization in Eosinophils

In this chapter, we will describe two different techniques used to trace storage and mobilization of intracellular granule-derived mediator proteins in eosinophils. The first is confocal laser scanning microscopy (CLSM) used to investigate immunofluorescence labeling in cyt ...

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Measurement of Metalloproteinases

Matrix metalloproteinases (MMPs) (1,2) comprise a family of over 20 matrix degrading enzymes believed to be essential for normal development and physiologic tissue remodeling and repair. Abnormal expression of metalloproteinases has been implicated in many destructive proce ...

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Immunocytochemistry and Immunohistochemistry

Immunohistochemistry allows the specific histochemical localization of many diverse classes of organic substances, including inflammatory markers ranging from biogenic amines to macromolecular cytokines and their receptors. Corresponding ligands and their recep ...

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Quantitative Analysis of Cyclooxygenase Metabolites of Arachidonic Acid

Metabolism of arachidonic acid results in a host of biologically active compounds with profound effects on airway inflammation (1). After activation of cellular phospholipases and release of free arachidonic acid, catalyzed insertion of oxygen occurs enzymatically via action of ...

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The Measurement of Cysteinyl Leukotrienes in Urine

The cysteinyl leukotrienes, comprising leukotriene (LT) C4 and its major metabolites LTD4 and LTE4, are inflammatory lipid mediators derived from metabolism of arachidonic acid by 5-lipoxygenase. These leukotrienes have received considerable attention for their potential ...

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